1. Field of the Invention
The present invention relates to a method of purifying nucleic acids using silver nanoparticles.
2. Description of the Related Art
The production of high purity double-strand plasmid DNAs, single-strand phage DNAs, chromosomal DNAs, and agarose gel-purified DNA fragments is very important in molecular biology. Ideal methods of purifying DNAs should be simple and can be performed rapidly and include little additional manipulation of samples. The DNAs obtained using such methods are ready for direct transformation, restriction enzyme analysis, ligation, or sequencing. Such methods are very attractive in the automated production of DNA samples, which is favored in research and diagnosis labs. Generally, the preparation of plasmid DNAs from crude alcohol precipitates is laborious. Plasmid DNAs are often produced using a CsCl gradient, gel filtration, ion exchange chromatography, RNAase, proteinase K, and repeated alcohol precipitation. These methods require considerable downstream sample preparation to remove CsCl and other salts, EtBr, and alcohol, etc. Further, small negatively charged cellular components can be precipitated together with DNAs. Thus, the DNAs may be contaminated to an undesirable degree.
Methods of purifying nucleic acids using solid phase materials are well known in the art. For example, U.S. Pat. No. 5,234,809 describes a method of purifying a nucleic acid using a solid phase material which can bind to the nucleic acid. Specifically, the method includes mixing a chaotropic material with a nucleic acid binding solid phase, separating the solid phase material with the nucleic acid bound thereto from the liquid, and washing the solid phase material-nucleic acid complexes. Examples of the chaotropic material include guanidinium thiocyanate (GuSCN), guanidine hydrochloride (GuHCl), sodium iodide (NaI), potassium iodide (KI), sodium thiocyanate (NaSCN), urea, and combinations thereof. Examples of the solid phase material include silica particles.
However, this method is considerably time-consuming, complicated, and unsuitable for lab-on-a-chip (LOC). Further, this method uses the chaotropic material as an essential component. If the chaotropic material is not used, nucleic acid cannot bind to the solid phase material. Since the chaotropic material is harmful to a human body, it must be carefully handled. In addition, the chaotropic material inhibits a subsequent process, such as PCR (polymerase chain reaction), and thus, it must be removed from the nucleic acid during or after purification of the nucleic acid.
Research has been conducted to develop a solid phase material having a large surface area for efficient binding with a nucleic acid. However, a method of purifying a nucleic acid using the solid phase material still requires many treatment processes and the use of chaotropic salts and is time-consuming.
A reversible immobilization method using a solid phase material has been described [Hawkins, et al., Nucleic Acids Res. 1995; 23:22]. This method is a simple method and easily automated. However, the application of this method to the detection of pathogens has not been described and this method is not suitable for the production of LOC.
There have been reported kits for producing single tube samples, which are available from GeneReleaser (manufactured by Bioventures), ReleaseIT (manufactured by CPG Inc.), and Lye-N-Go™ RCR Reagent (manufactured by Pierce). By using the kits, samples can be prepared in only two steps and PCR samples can be prepared within 10-15 minutes. However, areal-time PCR cannot be performed due to the use of a white polymer reagent and after cell lysis, reagents and a PCR mixture must be added, which is inconvenient and results in possible contamination of the samples.
The present inventors conducted research on a method of purifying a nucleic acid based on the general techniques and discovered that silver nanoparticles can bind to a molecule having a thiol group in a sample, and can then be removed from the sample using a SH-modified membrane.